Systems and methods for treatments of an eye with a photosensitizer

ABSTRACT

A formulation for an eye treatment includes a photosensitizer and a permeability enhancing composition. The permeability enhancing composition includes one or more permeability enhancers. The permeability enhancing composition has a hydrophilic and lipophilic balance increases a permeability of an area of the eye for the photosensitizer. The hydrophilic and lipophilic balance can be characterized by a Hydrophile-Lipophile Balance (HLB) number. For example, the area of the eye may include a corneal epithelium, the photosensitizer may include riboflavin, and the permeability enhancing composition may have a corresponding HLB number between approximately 12.6 and approximately 14.6.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/195,144, filed Jul. 21, 2015, U.S. Provisional Patent ApplicationNo. 62/255,452, filed Nov. 14, 2015, U.S. Provisional Patent ApplicationNo. 62/262,919, filed Dec. 4, 2015, and U.S. Provisional PatentApplication No. 62/263,598, filed Dec. 4, 2015, the contents of theseapplication being incorporated entirely herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present disclosure pertains to systems and methods for treating theeye, and more particularly, to systems and methods for delivering aphotosensitizer to regions of the eye for eye treatments.

Description of Related Art

Certain photosensitizers may be applied to the eye for eye treatments.For example, photosensitizers can generate cross-linking activity in thecornea. Cross-linking can treat disorders, such as keratoconus. Inparticular, keratoconus is a degenerative disorder of the eye in whichstructural changes within the cornea cause it to weaken and change to anabnormal conical shape. Cross-linking treatments can strengthen andstabilize areas weakened by keratoconus and prevent undesired shapechanges.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an example system that delivers a cross-linking agentand photoactivating light to a cornea of an eye in order to generatecross-linking of corneal collagen, according to aspects of the presentdisclosure.

FIGS. 2A-B illustrate a diagram for photochemical kinetic reactionsinvolving riboflavin and photoactivating light (e.g., ultraviolet A(UVA) light) applied during a corneal cross-linking treatment, accordingto aspects of the present disclosure.

FIGS. 3A-C illustrate graphs showing the correlation between modelvalues and experimental data for oxygen depletion experiments, where themodel values are based on a model of photochemical kinetic reactionsaccording to aspects of the present disclosure.

FIG. 4 illustrates a graph showing the correlation between model valuesand experimental data for non-linear optical microscopy fluorescenceexperiments, where the model values are based on a model ofphotochemical kinetic reactions according to aspects of the presentdisclosure.

FIGS. 5A-D illustrate graphs showing the correlation between modelvalues and experimental data for fluorescence data based on papaindigestion method experiments, where the model values are based on amodel of photochemical kinetic reactions according to aspects of thepresent disclosure.

FIGS. 6A-B illustrate graphs showing the correlation between modelvalues and experimental data for conical stromal demarcation lineexperiments, where the model values are based on a model ofphotochemical kinetic reactions according to aspects of the presentdisclosure.

FIGS. 7A-C illustrate graphs of cross-link profiles for treatments usingdifferent protocols, as generated by a model of photochemical kineticreactions according to aspects of the present disclosure.

FIGS. 8A-C illustrate graphs of cross-link profiles for treatments usingdifferent protocols, as generated by a model of photochemical kineticreactions, where the cross-link profiles are evaluated to determine thedepth for a demarcation line for each protocol according to aspects ofthe present disclosure.

FIGS. 9A-B illustrate graphs of demarcation depth versus dose ofphotoactivating light based on cross-link profiles for treatments usingdifferent protocols, as generated by a model of photochemical kineticreactions according to aspects of the present disclosure.

FIG. 10 illustrates a graph of cross-link profiles for treatments usingdifferent protocols as generated by a model of photochemical kineticreactions, where the cross-link profiles are evaluated to determine thedepth for a demarcation line for each protocol according to aspects ofthe present disclosure.

FIG. 11 illustrates the measurement of maximum keratometry (K_(max)) atsix and twelve months relative to a baseline for corneas that wereexperimentally treated according to the protocols employed for FIG. 10.

FIG. 12A illustrates a graph that plots, for the biomechanical stiffnessdepth determined for each protocol in FIG. 10, the experimental changeof K_(max) for months six and twelve corresponding to the respectiveprotocol, according to aspects of the present disclosure.

FIG. 12B illustrates a graph that plots, for the area above thedemarcation line for each protocol in FIG. 10, the experimental changeof K_(max) for months six and twelve corresponding to the respectiveprotocol, according to aspects of the present disclosure.

FIG. 13 illustrates an example system employing a model of photochemicalkinetic reactions according to aspects of the present disclosure.

FIG. 14 illustrates relative diffusivity values for differentformulations applied to corneas.

FIG. 15 illustrates relative fluorescence values for cross-linkedcorneas treated with different riboflavin formulations.

FIG. 16 illustrates different parameters for treating corneas withdifferent riboflavin formulations.

FIG. 17 illustrates relative fluorescence values for cross-linkedcorneas treated according to the parameters in FIG. 16.

FIGS. 18-20 illustrate relative fluorescence for cross-linked cornealflaps treated with different surfactants in riboflavin solution.

FIG. 21 illustrates relative fluorescence for cross-linked corneal flapstreated with a riboflavin solution that does not include benzalkoniumchloride (BAC), relative to a riboflavin solution that includes BAC as apermeability enhancer.

FIGS. 22-24 illustrate relative fluorescence for cross-linked cornealflaps treated with riboflavin solutions that include differentconcentrations of Polidocanol as a permeability enhancer, relative toother riboflavin solutions that include BAC as a permeability enhancerand to other riboflavin solutions without any permeability enhancer.

FIGS. 25-26 illustrate relative fluorescence for cross-linked cornealflaps treated with riboflavin solutions that include differentconcentrations of Polidocanol as a permeability enhancer or riboflavinsolutions that include different concentrations of Polidocanol as wellas Fe(II) as an additive, relative to other riboflavin solutions thatinclude BAC as a permeability enhancer.

FIG. 27 illustrates relative fluorescence of cross-linked flaps treatedwith one of two different surfactants or a combination of the twosurfactants.

FIG. 28A illustrates Brillouin modulus values measured at anterior,central, and posterior sections of corneas experimentally soaked inriboflavin for various durations and irradiated with UV light forvarious durations.

FIG. 28B illustrates the experimentally measured Brillouin modulusvalues and values calculated with a model of photochemical kineticreactions for various cross-linking treatments.

FIG. 29 illustrates concentration of cross-link concentration as well asa function of conical depth with a demarcation depth as well as thesecond derivative.

FIG. 30 illustrates another graph of data showing the correlation ofmodel values and experimental data for the depths of corneal stromaldemarcation lines for protocols described in fourteen separate studies.

FIG. 31 illustrates a graph of cross-link profiles (cross-linkconcentration as a function of conical depth) calculated by a model ofphotochemical kinetic reactions for various cross-linking treatments inextensiometry experiments.

FIG. 32 illustrates a correlation of extensiometry measurements andvalues calculated by the model for the various cross-linking treatmentsin the experiments of FIG. 31.

FIG. 33 illustrates how by a model of photochemical kinetic reactionsallows particular aspects of the photochemical process to be controlledor otherwise influenced to produce desired cross-linking activity.

FIG. 34 shows the effect of the various additives in FIG. 33 oncross-linking activity.

FIGS. 35-36 illustrate graphs of cross-link profiles for treatmentsemploying different protocols as generated by a model of photochemicalkinetic reactions.

FIG. 37 illustrates that the demarcation line depth may be affected byaspects of the riboflavin concentration, the use of thickening agent,illumination (UVA) device calibration, illumination (UVA) beam profile,and/or geographic factors.

FIG. 38 illustrates an example system employing a model of photochemicalkinetic reactions to provide treatment parameters for achieving desiredbiomechanical changes according to aspects of the present disclosure.

FIG. 39 an example method employing a model of photochemical kineticreactions to determine treatment parameters for achieving desiredbiomechanical changes according to aspects of the present disclosure.

SUMMARY

Aspects of the present disclosure relate to systems and methods fordelivering a photosensitizer to regions of the eye for eye treatments.In particular, the systems and methods employ formulations that enhancethe permeability of eye structures, such as the corneal epithelium, tofacilitate delivery of photosensitizers to desired areas.

According to aspects of the present disclosure, a formulation for an eyetreatment includes a photosensitizer and a permeability enhancingcomposition. The permeability enhancing composition includes one or morepermeability enhancers. The permeability enhancing composition has aHydrophile-Lipophile Balance (HLB) number indicating that thepermeability enhancing composition increases a permeability of an areaof the eye for the photosensitizer. In some cases, the permeabilityenhancing composition includes a plurality of permeability enhancers,where the HLB number for the permeability enhancing composition is equalto a sum of products multiplying a percentage of each permeabilityenhancer in the permeability enhancing composition and a HLB number forthe respective permeability enhancer. In other cases, the formulationfurther includes at least one additive selected from the groupconsisting of iron, copper, manganese, chromium, vanadium, aluminum,cobalt, mercury, cadmium, nickel, arsenic, 2,3-butanedione, and folicacid. For example, the at least one additive includes iron(II). Infurther cases, the area of the eye is a corneal epithelium. In yetfurther cases, the photosensitizer includes riboflavin and thepermeability enhancing composition has a HLB number betweenapproximately 12.6 and approximately 14.6. In alternative cases, thearea of the eye is an area infected by a pathogen.

According to further aspects of the present disclosure, a method fortreating an eye includes applying the formulation above to an area of aneye and photoactivating the photosensitizer by delivering a dose ofillumination to the area of the eye. In some cases, the method furtherincludes applying a concentration of oxygen to the cornea. In othercases, applying the formulation includes applying the formulation to anarea of the eye infected by a pathogen, whereby photoactivation of thephotosensitizer provides an antimicrobial effect on the area infected bythe pathogen. In alternative cases, applying the formulation includesapplying the formulation to a corneal epithelium.

According to other aspects of the present disclosure, a formulation fora corneal treatment includes a photosensitizer and a non-ionicsurfactant. The non-ionic surfactant includes a molecule having ahydrophilic and lipophilic balance that increases the permeability ofthe corneal epithelium for the photosensitizer. In some cases, thephotosensitizer includes riboflavin and the non-ionic surfactant has aHydrophile-Lipophile Balance (HLB) number between approximately 12.6 andapproximately 14.6. In other cases, the non-ionic surfactant includesPolyoxyethylene (9) lauryl ether. In yet other cases, the formulationincludes at least one additive including selected from the groupconsisting of iron, copper, manganese, chromium, vanadium, aluminum,cobalt, mercury, cadmium, nickel, arsenic, 2,3-butanedione, and folicacid. For example, the at least one additive includes iron(II).

DESCRIPTION

FIG. 1 illustrates an example treatment system 100 for generatingcross-linking of collagen in a cornea 2 of an eye 1. The treatmentsystem 100 includes an applicator 132 for applying a cross-linking agent130 to the cornea 2. In example embodiments, the applicator 132 may bean eye dropper, syringe, or the like that applies the photosensitizer130 as drops to the cornea 2. The cross-linking agent 130 may beprovided in a formulation that allows the cross-linking agent 130 topass through the corneal epithelium 2 a and to underlying regions in thecorneal stroma 2 b. Alternatively, the corneal epithelium 2 a may beremoved or otherwise incised to allow the cross-linking agent 130 to beapplied more directly to the underlying tissue.

The treatment system 100 includes an illumination system with a lightsource 110 and optical elements 112 for directing light to the cornea 2.The light causes photoactivation of the cross-linking agent 130 togenerate cross-linking activity in the cornea 2. For example, thecross-linking agent may include riboflavin and the photoactivating lightmay be ultraviolet A (UVA) (e.g., 365 nm) light. Alternatively, thephotoactivating light may have another wavelength, such as a visiblewavelength (e.g., 452 nm). As described further below, cornealcross-linking improves corneal strength by creating chemical bondswithin the corneal tissue according to a system of photochemical kineticreactions. For instance, riboflavin and the photoactivating light areapplied to stabilize and/or strengthen corneal tissue to addressdiseases such as keratoconus or post-LASIK ectasia.

The treatment system 100 includes one or more controllers 120 thatcontrol aspects of the system 100, including the light source 110 and/orthe optical elements 112. In an implementation, the cornea 2 can be morebroadly treated with the cross-linking agent 130 (e.g., with an eyedropper, syringe, etc.), and the photoactivating light from the lightsource 110 can be selectively directed to regions of the treated cornea2 according to a particular pattern.

The optical elements 112 may include one or more mirrors or lenses fordirecting and focusing the photoactivating light emitted by the lightsource 110 to a particular pattern on the cornea 2. The optical elements112 may further include filters for partially blocking wavelengths oflight emitted by the light source 110 and for selecting particularwavelengths of light to be directed to the cornea 2 for activating thecross-linking agent 130. In addition, the optical elements 112 mayinclude one or more beam splitters for dividing a beam of light emittedby the light source 110, and may include one or more heat sinks forabsorbing light emitted by the light source 110. The optical elements112 may also accurately and precisely focus the photo-activating lightto particular focal planes within the cornea 2, e.g., at a particulardepths in the underlying region 2 b where cross-linking activity isdesired.

Moreover, specific regimes of the photoactivating light can be modulatedto achieve a desired degree of cross-linking in the selected regions ofthe cornea 2. The one or more controllers 120 may be used to control theoperation of the light source 110 and/or the optical elements 112 toprecisely deliver the photoactivating light according to any combinationof: wavelength, bandwidth, intensity, power, location, depth ofpenetration, and/or duration of treatment (the duration of the exposurecycle, the dark cycle, and the ratio of the exposure cycle to the darkcycle duration).

The parameters for photoactivation of the cross-linking agent 130 can beadjusted, for example, to reduce the amount of time required to achievethe desired cross-linking. In an example implementation, the time can bereduced from minutes to seconds. While some configurations may apply thephotoactivating light at an irradiance of 5 mW/cm², larger irradiance ofthe photoactivating light, e.g., multiples of 5 mW/cm², can be appliedto reduce the time required to achieve the desired cross-linking. Thetotal dose of energy absorbed in the cornea 2 can be described as aneffective dose, which is an amount of energy absorbed through an area ofthe corneal epithelium 2 a. For example the effective dose for a regionof the corneal surface 2A can be, for example, 5 J/cm², or as high as 20J/cm² or 30 J/cm². The effective dose described can be delivered from asingle application of energy, or from repeated applications of energy.

The optical elements 112 of the treatment system 100 may include adigital micro-mirror device (DMD) to modulate the application ofphotoactivating light spatially and temporally. Using DMD technology,the photoactivating light from the light source 110 is projected in aprecise spatial pattern that is created by microscopically small mirrorslaid out in a matrix on a semiconductor chip. Each mirror represents oneor more pixels in the pattern of projected light. With the DMD one canperform topography guided cross-linking. The control of the DMDaccording to topography may employ several different spatial andtemporal irradiance and dose profiles. These spatial and temporal doseprofiles may be created using continuous wave illumination but may alsobe modulated via pulsed illumination by pulsing the illumination sourceunder varying frequency and duty cycle regimes as described above.Alternatively, the DMD can modulate different frequencies and dutycycles on a pixel by pixel basis to give ultimate flexibility usingcontinuous wave illumination. Or alternatively, both pulsed illuminationand modulated DMD frequency and duty cycle combinations may be combined.This allows for specific amounts of spatially determined cornealcross-linking. This spatially determined cross-linking may be combinedwith dosimetry, interferometry, optical coherence tomography (OCT),corneal topography, etc., for pre-treatment planning and/or real-timemonitoring and modulation of corneal cross-linking during treatment.Additionally, pre-clinical patient information may be combined withfinite element biomechanical computer modeling to create patientspecific pre-treatment plans.

To control aspects of the delivery of the photoactivating light,embodiments may also employ aspects of multiphoton excitationmicroscopy. In particular, rather than delivering a single photon of aparticular wavelength to the cornea 2, the treatment system 100 maydeliver multiple photons of longer wavelengths, i.e., lower energy, thatcombine to initiate the cross-linking. Advantageously, longerwavelengths are scattered within the cornea 2 to a lesser degree thanshorter wavelengths, which allows longer wavelengths of light topenetrate the cornea 2 more efficiently than shorter wavelength light.Shielding effects of incident irradiation at deeper depths within thecornea are also reduced over conventional short wavelength illuminationsince the absorption of the light by the photosensitizer is much less atthe longer wavelengths. This allows for enhanced control over depthspecific cross-linking. For example, in some embodiments, two photonsmay be employed, where each photon carries approximately half the energynecessary to excite the molecules in the cross-linking agent 130 togenerate the photochemical kinetic reactions described further below.When a cross-linking agent molecule simultaneously absorbs both photons,it absorbs enough energy to release reactive radicals in the cornealtissue. Embodiments may also utilize lower energy photons such that across-linking agent molecule must simultaneously absorb, for example,three, four, or five, photons to release a reactive radical. Theprobability of the near-simultaneous absorption of multiple photons islow, so a high flux of excitation photons may be required, and the highflux may be delivered through a femtosecond laser.

A large number of conditions and parameters affect the cross-linking ofcorneal collagen with the cross-linking agent 130. For example, when thecross-linking agent 130 is riboflavin and the photoactivating light isUVA light, the irradiance and the dose both affect the amount and therate of cross-linking. The UVA light may be applied continuously(continuous wave (CW)) or as pulsed light, and this selection has aneffect on the amount, the rate, and the extent of cross-linking.

If the UVA light is applied as pulsed light, the duration of theexposure cycle, the dark cycle, and the ratio of the exposure cycle tothe dark cycle duration have an effect on the resulting cornealstiffening. Pulsed light illumination can be used to create greater orlesser stiffening of corneal tissue than may be achieved with continuouswave illumination for the same amount or dose of energy delivered. Lightpulses of suitable length and frequency may be used to achieve moreoptimal chemical amplification. For pulsed light treatment, the on/offduty cycle may be between approximately 1000/1 to approximately 1/1000;the irradiance may be between approximately 1 mW/cm² to approximately1000 mW/cm² average irradiance, and the pulse rate may be betweenapproximately 0.01 HZ to approximately 1000 Hz or between approximately1000 Hz to approximately 100,000 Hz.

The treatment system 100 may generate pulsed light by employing a DMD,electronically turning the light source 110 on and off, and/or using amechanical or opto-electronic (e.g., Pockels cells) shutter ormechanical chopper or rotating aperture. Because of the pixel specificmodulation capabilities of the DMD and the subsequent stiffnessimpartment based on the modulated frequency, duty cycle, irradiance anddose delivered to the cornea, complex biomechanical stiffness patternsmay be imparted to the cornea to allow for various amounts of refractivecorrection. These refractive corrections, for example, may involvecombinations of myopia, hyperopia, astigmatism, irregular astigmatism,presbyopia and complex corneal refractive surface corrections because ofophthalmic conditions such as keratoconus, pellucid marginal disease,post-lasik ectasia, and other conditions of corneal biomechanicalalteration/degeneration, etc. A specific advantage of the DMD system andmethod is that it allows for randomized asynchronous pulsed topographicpatterning, creating a non-periodic and uniformly appearing illuminationwhich eliminates the possibility for triggering photosensitive epilepticseizures or flicker vertigo for pulsed frequencies between 2 Hz and 84Hz.

Although example embodiments may employ stepwise on/off pulsed lightfunctions, it is understood that other functions for applying light tothe cornea may be employed to achieve similar effects. For example,light may be applied to the cornea according to a sinusoidal function,sawtooth function, or other complex functions or curves, or anycombination of functions or curves. Indeed, it is understood that thefunction may be substantially stepwise where there may be more gradualtransitions between on/off values. In addition, it is understood thatirradiance does not have to decrease down to a value of zero during theoff cycle, and may be above zero during the off cycle. Desired effectsmay be achieved by applying light to the cornea according to a curvevarying irradiance between two or more values.

Examples of systems and methods for delivering photoactivating light aredescribed, for example, in U.S. Patent Application Publication No.2011/0237999, filed Mar. 18, 2011 and titled “Systems and Methods forApplying and Monitoring Eye Therapy,” U.S. Patent ApplicationPublication No. 2012/0215155, filed Apr. 3, 2012 and titled “Systems andMethods for Applying and Monitoring Eye Therapy,” and U.S. PatentApplication Publication No. 2013/0245536, filed Mar. 15, 2013 and titled“Systems and Methods for Corneal Cross-Linking with Pulsed Light,” thecontents of these applications being incorporated entirely herein byreference.

The addition of oxygen also affects the amount of corneal stiffening. Inhuman tissue, O₂ content is very low compared to the atmosphere. Therate of cross-linking in the cornea, however, is related to theconcentration of O₂ when it is irradiated with photoactivating light.Therefore, it may be advantageous to increase or decrease theconcentration of O₂ actively during irradiation to control the rate ofcross-linking until a desired amount of cross-linking is achieved.Oxygen may be applied during the cross-linking treatments in a number ofdifferent ways. One approach involves supersaturating the riboflavinwith O₂. Thus, when the riboflavin is applied to the eye, a higherconcentration of O₂ is delivered directly into the cornea with theriboflavin and affects the reactions involving O₂ when the riboflavin isexposed to the photoactivating light. According to another approach, asteady state of O₂ (at a selected concentration) may be maintained atthe surface of the cornea to expose the cornea to a selected amount ofO₂ and cause O₂ to enter the cornea. As shown in FIG. 1, for instance,the treatment system 100 also includes an oxygen source 140 and anoxygen delivery device 142 that optionally delivers oxygen at a selectedconcentration to the cornea 2. Example systems and methods for applyingoxygen during cross-linking treatments are described, for example, inU.S. Pat. No. 8,574,277, filed Oct. 21, 2010 and titled “Eye Therapy,”U.S. Patent Application Publication No. 2013/0060187, filed Oct. 31,2012 and titled “Systems and Methods for Corneal Cross-Linking withPulsed Light,” the contents of these applications being incorporatedentirely herein by reference.

In general, the structure of the cornea includes five layers. From theouter surface of the eye inward, these are: (1) epithelium, (2) Bowman'slayer, (3) stroma, (4) Descemet's membrane, and (5) endothelium. Duringexample cross-linking treatments, the stroma is treated with riboflavin,a photosensitizer, and ultraviolet (UV) light is delivered to the corneato activate the riboflavin in the stroma. Upon absorbing UV radiation,riboflavin undergoes a reaction with oxygen in which reactive oxygenspecies and other radicals are produced. These reactive oxygen speciesand other radicals further interact with the collagen fibrils to inducecovalent bonds that bind together amino acids of the collagen fibrils,thereby cross-linking the fibrils. The photo-oxidative induction ofcollagen cross-linking enhances the biomechanical strength of thestroma, and can provide therapeutic benefits for certain ophthalmicconditions, such as keratoconus, or generate refractive changes tocorrect myopia, hyperopia and/or astigmatism.

As the outer-most barrier of the cornea, the epithelium functions toregulate nutrients, including oxygen, that are admitted into the stromaltissue from the tear film. This regulation is carried out via theepithelium's physiological “pumps” that are driven by osmotic pressureacross the epithelium due to differential concentrations ofbarrier-permeable solutes on either side of the epithelium. Whenhealthy, certain nutrients in the tear film that become depleted withinthe stroma can permeate the epithelium via osmotic pressure to resupplythe stroma. However, while oxygen and some other small moleculenutrients can reach the stroma according to this mechanism, certainphotosensitizers cannot pass through the epithelium.

Riboflavin, for example, is a relatively large, hydrophilic moleculethat cannot penetrate the tight junctions of the epithelium. Theepithelium slows the amount of riboflavin that can penetrate the stroma.Thus, a variety of approaches have been employed to overcome lowriboflavin diffusivity and deliver sufficient concentrations ofriboflavin to the stroma for performing corneal cross-linkingtreatments. According to one approach, the epithelium is removed(epithelium debridement) before a riboflavin solution is applieddirectly to the stroma. Although removing the epithelium allowsriboflavin to reach the stroma, the approach is associated with patientdiscomfort, risks of infection, and other possible complications.

Meanwhile, other approaches avoid epithelial debridement. For example,riboflavin may be provided in a formulation that allows thecross-linking agent to pass through the epithelium. Such formulationsare described, for example, in U.S. Patent Application Publication No.2010/0286156, filed on May 6, 2009 and titled “Collyrium for theTreatment of Conical Cornea with Cross-Linking Trans-EpithelialTechnique, and in U.S. Patent Application Publication No. 2013/0267528,filed on Jan. 4, 2013 and titled “Trans-Epithelial Osmotic Collyrium forthe Treatment of Keratoconus,” the contents of these applications beingincorporated entirely herein by reference. In particular, someriboflavin formulations include ionic agents, such as benzalkoniumchloride (BAC), with a specific osmolarity of sodium chloride (NaCl).Although such formulations may enhance permeability of the epithelium,they are disadvantageously corrosive to the epithelium.

Additionally or alternatively, another solution and/or mechanical forcesmay be applied to enhance the permeability of the epithelium and allowthe riboflavin to pass more easily through the epithelium. Examples ofapproaches for enhancing or otherwise controlling the delivery of across-linking agent to the underlying regions of the cornea aredescribed, for example, in U.S. Patent Application Publication No.2011/0288466, filed Apr. 13, 2011 and titled “Systems and Methods forActivating Cross-Linking in an Eye,” and U.S. Patent ApplicationPublication No. 2012/0289886, filed May 18, 2012 and titled “ControlledApplication of Cross-Linking Agent,” the contents of these applicationsbeing incorporated entirely herein by reference.

The present disclosure teaches the use of another class of riboflavinformulations. Advantageously, such formulations enhance the permeabilityof the epithelium sufficiently to allow relatively large hydrophilicriboflavin molecules (or Flavin mononucleotide (FMN), orriboflavin-5′-phosphate, molecules) to pass through the epitheliumwithout debridement, but the permeability is not enhanced to a pointwhere the epithelium becomes damaged. To enhance permeability, suchformulations employ a non-ionic agent that is chosen using theHydrophile-Lipophile Balance (HLB) system.

The HLB of a permeability enhancer indicates the balance of hydrophilicand lipophilic groups in the molecular structure of the enhancer.Permeability enhancers (or emulsifiers) for the epithelium include amolecule which has both hydrophilic and lipophilic groups. Moleculeswith HLB number below 9 are considered lipophilic and those above 11 ashydrophilic. Molecules with HLB number between 9 and 11 areintermediate.

For the corneal epithelium, a HLB number that is too great or too smalldoes not help the passage of a photosensitizer through the epithelium. Aspecific HLB range enhances movement of a photosensitizer through theepithelium. Thus, aspects of the present disclosure employ non-ionicagents that have a hydrophilic/lipophilic balance to achieve optimizeddiffusivity through the epithelium and the stroma. Advantageously,non-ionic agents are also less corrosive and damaging to the epitheliumthan ionic agents, such as BAC.

For riboflavin, the HLB range for more effective permeability enhancershas been experimentally determined by the inventors to be betweenapproximately 12.6 and approximately 14.6. A class of permeabilityenhancers includes various forms of polyethylene glycol (PEG) withdifferent aliphatic chain lengths. According to example embodiments,some riboflavin formulations include specific concentrations ofPolidocanol (Polyoxyethylene (9) lauryl ether), which has a HLB numberof approximately 13.6.

To calculate the HLB for molecules or combinations of molecules wherethe hydrophilic portion consists of ethylene oxide only, the formula is:HLB=E/5, where E=weight percentage oxyethylene content.

In general, the HLB range for enhancers that achieve more effectivepermeability may vary according to different aspects of the formulation.In particular, the HLB range for more optimal enhancers may varyaccording to the photosensitizer employed in the formulation. Forinstance, more optimal permeability might be achieved for otherphotosensitizers, such as Rose Bengal, by employing enhancers in a HLBrange that is different from that for riboflavin (e.g., HLB ofapproximately 12.6 to approximately 14.6).

Furthermore, the formulation may include other additives that may affectthe HLB range for more optimal enhancers. For instance, riboflavinformulations may also include iron ions, such as Fe(II). Additives thatmay be included in photosensitizer formulations are described, forexample, in U.S. Patent Application Publication No. 2014/0343480, filedMay 19, 2014 and titled “Systems, Methods, and Compositions forCross-linking,” and U.S. Provisional Patent Application No. 62/086,572,filed Dec. 2, 2014 and titled “Systems, Methods, and Compositions forCross-linking,” the contents of these applications being incorporatedentirely herein by reference. Other additives, for instance, includecopper, manganese, chromium, vanadium, aluminum, cobalt, mercury,cadmium, nickel, arsenic, 2,3-butanedione, and folic acid.

Additionally, several permeability enhancers may be combined to achievea specific HLB that achieves more effective permeability for theepithelium. These may be calculated by taking the percentage of eachenhancer, multiplying it by its HLB number, and then summing theresults. For instance, in a formulation including 30% enhancer A with aHLB number of approximately 14, 50% enhancer B with a HLB number ofapproximately 6, and 20% enhancer C with a HLB number of approximately14, the estimated HLB number can be calculated as:30%×HLB 14 for A=4.2;50%×HLB 6 for B=3.0;20%×HLB 14 for C=2.8;Estimated HLB number for combination of A+B+C=4.2+3.0+2.8=10.0

Thus, two or more enhancers may be combined to achieve a very specificHLB number, where a single enhancer may provide less optimalpermeability. Additionally, combining different enhancers might offerother desirable properties of the final formulation with regard tosolubility, viscosity, stability or some other desirable attribute.

Study 1

In a study, a Franz cell was employed to measure diffusivity ofriboflavin formulations containing BAC or a non-ionic agent in porcineeyes with or without an epithelia of approximately 100 μm. FIG. 14illustrates a graph of diffusivity values for the formulations in thisstudy. Column A represents the application of a saline solution of 0.1%riboflavin to porcine eyes without epithelia (epi-off). Column Brepresents the application of a 0.22% riboflavin solution with BAC toporcine eyes epi-off. Column C represents the application of a 0.22%riboflavin solution with a non-ionic agent to porcine eyes withepithelia (epi-on). Column D represents the application of a 0.22%riboflavin solution with BAC to porcine eyes epi-on. Column E representsthe application of a saline solution of 0.1% riboflavin to porcine eyesepi-on. The results indicates the formulation with the non-ionic agentformulation achieved faster diffusion than the ionic formulation withBAC. Furthermore, the diffusivity of the formulation with the non-ionicagent applied epi-on is similar to the diffusivity for the formulationsapplied epi-off. Thus, a sufficient hydrophilic/lipophilic balance wasachieved with the non-ionic agent.

Study 2

In a study, porcine eyes shipped overnight on ice from an abattoir(SiouxPreme, Sioux City, Iowa) were cleaned and soaked for 20 minutes inan incubator set at 37° C. with a 0.22% riboflavin solution with BAC ora 0.22% riboflavin solution with a non-ionic agent. The corneas hadepithelia of approximately 100 μm. The epithelia of the corneas wereremoved after the respective soaks and prior to pan-corneal irradiationwith UVA light. The treatment protocol employed applying pulsed UVAlight (1 second on; 1 second off) at an irradiation of 30 mW/cm² and fora dose of 7.2 J/cm², while the corneas were exposed to 100%concentration of oxygen gas. 200 μm corneal flaps were cut using afemtosecond laser. The extent of the cross-linking in the corneas wasevaluated on the basis of fluorimetric analysis (excitation wave 365 nm,emission wave 450 nm) after collagen solubilization with papain.

FIG. 15 illustrates the fluorescence values for the formulations in thisstudy, indicating the extent of cross-linking activity. Column Arepresents the fluorescence of the corneas treated with the 0.22%riboflavin solution with BAC. Column B represents the fluorescence ofthe corneas treated with the 0.22% riboflavin solution with thenon-ionic agent. The results indicate that a smaller concentration ofriboflavin passed through the 100 μm epithelium and the cross-linking isriboflavin-limited when the BAC formulation was employed. In general,epi-on cross-linking requires a sufficient riboflavin concentration inthe stroma to achieve greater cross-linking efficiency.

Study 3

In a study, porcine eyes were treated according to the parametersindicated in FIG. 16. To provide a control, porcine eyes were soakedepi-off with 0.1% riboflavin solution for 20 minutes and then irradiatedwith continuous wave UVA light with an irradiance of 3 mW/cm² for a doseof 5.4 J/cm² while exposed to ambient air. To provide another control,porcine eyes were soaked epi-off with 0.22% riboflavin solution for 20minutes and then irradiated with continuous wave UVA light with anirradiance of 30 mW/cm² for a dose of 7.2 J/cm² while exposed to ambientair. Additionally, porcine eyes were soaked epi-on with 0.22% riboflavinsolution with BAC for 20 minutes and then irradiated with continuouswave UVA light with an irradiance of 30 mW/cm² for a dose of 7.2 J/cm²while exposed to ambient air. Porcine eyes were soaked epi-on with 0.22%riboflavin solution with a non-ionic agent for 20 minutes and thenirradiated with continuous wave UVA light with an irradiance of 30mW/cm² for a dose of 7.2 J/cm² while exposed to ambient air. Porcineeyes were soaked epi-on with 0.22% riboflavin solution with BAC for 20minutes and then irradiated with pulsed UVA light with an irradiance of30 mW/cm² for a dose of 7.2 J/cm² while exposed to 100% oxygen. Porcineeyes were soaked epi-on with 0.22% riboflavin solution with thenon-ionic agent for 20 minutes and then irradiated with pulsed UVA lightwith an irradiance of 30 mW/cm² for a dose of 7.2 J/cm² while exposed to100% oxygen.

The extent of the cross-linking in the corneas was evaluated on thebasis of fluorimetric analysis. FIG. 17 shows the fluorescence valuesfor the formulations in this study, indicating the extent ofcross-linking activity. Columns A-F in FIG. 17 represent the resultscorresponding to the experimental parameters provided in respective rowsA-F in FIG. 16. Columns C and D indicate that the cross-linking with the0.22% riboflavin solution with BAC or the 0.22% riboflavin solution withthe non-ionic agent was oxygen-limited. Columns E and F indicate thatthe cross-linking with the 0.22% riboflavin solution with BAC isriboflavin limited when compared to the cross-linking with the 0.22%riboflavin solution with the non-ionic agent. In addition, Columns E andF indicate that absorption by riboflavin in the saturated epithelium isnot a significant factor when oxygen is applied.

In view of the foregoing, the diffusivity of riboflavin and the initialstromal concentration of riboflavin affects the extent of cross-linkingactivity. The results from the formulations including the non-ionicagent indicate that hydrophilic-lipophilic properties are a factor,allowing riboflavin to penetrate the epithelium and diffuse into thecorneal hydrophilic stroma in quantities and duration appropriate for aclinical application.

Oxygen is a factor in efficient trans-epithelial (epi on) cross-linking.The results of the study show that the application of oxygen with thenon-ionic agent provides cross-linking efficiencies similar to standardepi-off cross-linking.

Less oxygen may generally be available in the stroma due to theepithelial thickness as it relates to Fick's law of diffusion and due tophoto-induced oxygen consumption in the epithelium. Thus, this studyshows that the additional application of oxygen can enhance epi oncross-linking efficiency.

In addition, the absorption of UVA light by riboflavin-saturatedepithelium may reduce photon efficiency. However, the results of thisstudy indicate that, when oxygen is also applied, the absorption byriboflavin-saturated epithelium is not a predominate factor.

As described above, some riboflavin formulations include specificconcentrations of Polidocanol to enhance permeability of the cornealepithelium. Advantageously, the concentrations of Polidocanol do notcause damage to the epithelium. Such riboflavin solutions may alsoinclude additives such as Fe(II).

Polidocanol and optionally additives can be employed in combination withother cross-linking techniques as described above to enhance delivery ofriboflavin through the epithelium and achieve the desired amount ofcross-linking activity. For instance, the riboflavin formulations withPolidocanol and optional additives can be applied with oxygen.Furthermore, the riboflavin solutions can be employed with differentapproaches for delivering photoactivating illumination (pulsedillumination, illumination of different patterns, etc.).

Study 4

To identify a new trans-epithelial formulation containing riboflavin, astudy was conducted to test riboflavin formulations with Polidocanol asa less toxic and more efficient substitute for riboflavin formulationwith benzalkonium chloride (BAC).

Pig eyes shipped overnight on ice from an abattoir (SiouxPreme, SiouxCity, Iowa) were rinsed in saline. The eyes with intact epithelium weresoaked with one of the test solutions below for 20 minutes in anincubator set at 37° C. by using a rubber ring to hold the solution ontop.

For a Group A of the pig eyes, the following riboflavin formulationswere used:

-   -   (a1) 0.25% riboflavin solution containing BAC (PARACEL™, Avedro,        Inc., Waltham, Mass.);    -   (a2) 0.1% w.v. riboflavin solution containing saline (PHOTREXA        ZD™, Avedro, Inc., Waltham, Mass.) with added riboflavin to        match the riboflavin content (0.25%) in solution (a1);    -   (a3) solution (a2) with 1% Polidocanol;    -   (a4) solution (a2) with 5% Polidocanol; and    -   (a5) solution (a2) with 10% Polidocanol.

The epitheliums of eyes in Group A were removed with a dull blade afterthe eyes were soaked in one of the solutions and irradiatedpan-corneally on air with a top hat beam (3% root mean square) for 4minutes with 365-nm light source (UV LED NCSU033B[T]; Nichia Co.,Tokushima, Japan) at a chosen irradiance of 30 mW/cm² which was measuredwith a power sensor (model PD-300-UV; Ophir, Inc., Jerusalem, Israel) atthe corneal surface. Corneal flaps (approximately 200 μm thick) wereexcised from the eyes with aid of an Intralase femtosecond laser (AbbotMedical Optics, Santa Ana, Calif.). The average thickness of the cornealflaps was calculated as a difference between the measurements before andafter the excision from the eyes with an ultrasonic Pachymeter (DGHTechnology, Exton, Pa.). The flaps were washed with distilled water anddried in a vacuum until the weight change became less than 10% (Rotaryvane vacuum pump RV3 A652-01-903, BOC Edwards, West Sussex, UK). Eachflap was digested for 2.5 h at 65° C. with 2.5 units/ml of papain (fromPapaya latex, Sigma) in 1 ml of papain buffer [BBBS (pH 7.0-7.2), 2 mML-cysteine and 2 mM EDTA]. Papain digests were diluted 0.5 times with1×BBBS and fluorescence of the solutions was measured with excitation of360 nm in a QM-40 Spectrofluorometer (Photon Technology Int., London,Ontario, Canada). The fluorescence of the papain buffer was taken intoaccount by measuring fluorescence in the absence of tissue andsubtracting this value from the fluorescence of the samples.

For a Group B of the pig eyes, the following riboflavin solutions wereused:

-   -   (b1) 0.25% riboflavin solution containing BAC (PARACEL™);    -   (b2) 0.22% riboflavin solution containing saline (VIBEX XTRA™,        Avedro, Inc., Waltham, Mass.) with 1% Polidocanol;    -   (b3) 0.22% riboflavin solution containing saline (VIBEX XTRA™)        with 5% Polidocanol; and    -   (b4) 0.22% riboflavin solution containing saline (VIBEX XTRA™).

The epitheliums of eyes in Group B were not removed after soaking in oneof the solutions and the surfaces were briefly rinsed with a salinebuffer before irradiation. The epitheliums were removed after theirradiation. Conditions used for the irradiation and the followingtreatment of the eyes were the same as for Group A.

For a Group C of the pig eyes, the following riboflavin solutions wereused:

-   -   (c1) 0.25% riboflavin solution containing BAC (PARACEL™);    -   (c2) 0.22% riboflavin solution containing saline (VIBEX XTRA™)        with 1% Polidocanol; and    -   (c3) 0.22% riboflavin solution containing saline (VIBEX XTRA™)        with 3% Polidocanol.

The epitheliums of eyes in Group C were not removed after soaking in oneof the solutions and the surfaces were briefly rinsed with a salinebuffer before irradiation. The eyes were placed in a beaker with anoxygen stream for 2 minutes in the incubation chamber prior toirradiation. Corneas were pan-corneally irradiated with irradiance of 30mW/cm², pulsed 1 sec on: 1 sec off for a total time of 8 min (7.2 J).The eyes were exposed to oxygen during all time of the treatment. Theepithelium were removed from the cornea after the irradiation with adull blade. Corneal flaps (approximately 200 μm thick) were excised fromthe eyes with aid of Intralase femtosecond laser and the followingtreatment of the flaps was the same as for the Groups A and B.

For a Group D and a Group E of the pig eyes, the following riboflavinsolutions were used:

-   -   (d1) 0.25% riboflavin solution containing BAC (PARACEL™);    -   (d2) 0.22% riboflavin solution containing saline (VIBEX XTRA™)        with 1% Polidocanol;    -   (d3) 0.22% riboflavin solution containing saline (VIBEX XTRA™)        with 1% Polidocanol and 2.5 mM Fe(II).    -   (d4) 0.22% riboflavin solution containing saline (VIBEX XTRA™)        with 3% Polidocanol; and    -   (d5) 0.22% riboflavin solution containing saline (VIBEX XTRA™)        with 3% Polidocanol and 2.5 mM Fe(II).

For Group D, the experimental procedure (including the irradiation,oxygen exposure, and the cutting of the flaps) was the same as for GroupC.

The epitheliums of eyes in Group E were removed after soaking in one ofthe solutions for 20 min. The eyes then were placed in a beaker withoxygen stream for 2 minutes in the incubation chamber prior toirradiation. Corneas were pan-corneally irradiated with irradiance of 30mW/cm², pulsed 1 sec on: 1 sec off for total time of 8 min (7.2 J). Theeyes were exposed to oxygen during all time of the treatment. Cornealflaps (approximately 200 μm thick) were excised from the eyes with aidof Intralase femtosecond laser and the following treatment of the flapswas the same as for the Groups A and B.

FIGS. 18-26 illustrate the cross-linking activity induced in Groups A-Eby various riboflavin solutions. The cross-linking activity was measuredas a ratio of fluorescence for the treated sample (F) to fluorescencefor an untreated control (Fo), where emissions were recorded at awavelength of 450 nm. Such measurement of cross-linking activity isdescribed, for example, in U.S. Pat. No. 9,020,580, filed Jun. 4, 2012and titled “Systems and Methods for Monitoring Time Based Photo ActiveAgent Delivery or Photo Active Marker Presence,” the contents of whichare incorporated entirely herein by reference.

FIGS. 18 and 19 illustrate relative fluorescence for cross-linkedcorneal flaps treated with different surfactants in 0.22% riboflavinsolution containing saline (VIBEX XTRA™) applied topically to pig eyeswith intact epithelium for 20 min, after which the epithelium were thenremoved and the eyes were irradiated with 30 mW/cm² for 4 min. Theseresults are presented in relation to corneal flaps treated with 0.25%riboflavin solution containing BAC (PARACEL™) under the same proceduralconditions.

FIG. 20 illustrates relative fluorescence for cross-linked corneal flapsafter using 1% solutions of different surfactants in 0.22% riboflavinsolution containing saline (VIBEX XTRA™). These results are presented inrelation to the fluorescence from corneal flaps treated with only 0.25%riboflavin solution containing BAC (PARACEL™) in the same proceduralconditions. FIGS. 3-5 also show the HLB numbers for the surfactants,e.g., Polidocanol has an HLB number of 13.6.

For Group A, FIG. 21 illustrates relative fluorescence for cross-linkedconical flaps treated with solution (a2) which does not include BACrelative to solution (a1) which includes BAC. Meanwhile, FIG. 22illustrates relative fluorescence of cross-linked conical flaps treatedwith solutions (a3), (a4), and (a5) which include differentconcentrations of Polidocanol. These results are presented relative toconical flaps treated with solution (a1) which includes BAC and cornealflaps with no epithelium treated with solution (a2) which does notinclude Polidocanol or BAC.

For Group B, FIG. 23 illustrates relative fluorescence for cross-linkedcorneal flaps treated with solutions (b2) and (b3) which include 1% and5% concentrations of Polidocanol respectively. These results arepresented relative to corneal flaps treated with solution (b1) whichincludes BAC and corneal flaps treated with solution (b4) which does notinclude Polidocanol or BAC.

For Group C, FIG. 24 illustrates relative fluorescence of cross-linkedconical flaps treated with solutions (c2) and (c3) which include 1% and3% concentrations of Polidocanol respectively. These results arepresented relative to conical flaps treated with solution (c1) whichincludes BAC.

FIG. 25 for Group D and FIG. 11 for Group E illustrate relativefluorescence of cross-linked flaps treated with solutions (d2) and (d4)which include 1% and 3% concentrations of Polidocanol respectively andwith solutions (d3) and (d5) which include 2.5 mM iron(II) as well as 1%and 3% concentrations of Polidocanol respectively. These results arepresented relative to conical flaps treated with solution (d1) whichincludes BAC. As described above, the epitheliums in Group D were notremoved after soaking in the solutions, while the epitheliums in Group Ewere removed after soaking in the solutions.

As the results of the study show, the inventors have identifiedPolidocanol as a non-ionic surfactant that is more effective than manyother surfactants for enhancing permeability and generatingcross-linking activity. Although the use of BAC in riboflavin solutionsmay help riboflavin to pass through the epithelium, Polidocanol is farmore effective and efficient than BAC in enhancing permeability in theepithelium and generating cross-linking activity. Advantageously,non-ionic agents, such as Polidocanol, are less corrosive and damagingto the epithelium than BAC.

Study 5

As described above, several permeability enhancers may be combined toachieve a specific HLB that achieves more optimal permeability for theepithelium. A study was conducted to test combinations of surfactantswith different HLB numbers.

Intact epithelium were soaked for 20 min using one of the followingsolutions:

-   -   (e1) 0.25% riboflavin solution containing BAC (PARACEL™);    -   (e2) 0.22% riboflavin solution containing saline (VIBEX XTRA™)        with 1% IGEPAL CO-630;    -   (e3) 0.22% riboflavin solution containing saline (VIBEX XTRA™)        with 1% IGEPAL CO-720; and    -   (e4) 0.22% riboflavin solution containing saline (VIBEX XTRA™)        with 1% mixture of IGEPAL CO-630 with IGEPAL CO-720 (1:1 ratio).

The epitheliums of the eyes were removed and the eyes were irradiatedwith 30 mW/cm² for 4 min continuously on air. Corneal flaps withthickness of 200 μm were cut and the papain digestion and fluorescenceanalysis was conducted as previously described above.

FIG. 27 illustrates relative fluorescence of the cross-linked flapstreated with one of two different surfactants or a combination of thetwo surfactants. The cross-linking activity was measured as a ratio offluorescence for the respective treated sample (F) to fluorescence for asample treated with solution (e1) (Fparacel), where emissions wererecorded at a wavelength of 450 nm.

The surfactant IGEPAL CO-630 has a HLB number of 13 and the surfactantIGEPAL CO-720 has a HLB number of 14, the 1:1 mixture has a HLB numberof 13.5. As FIG. 12 shows, the mixture of the surfactants facilitatesriboflavin permeation through the corneal epithelium more effectivelythan the surfactants employed individually.

Although the examples described herein may relate to the use ofriboflavin and Polidocanol as a permeability enhancer for cornealcross-linking treatments, it is understood that other photosensitizersand/or other permeability enhancers (e.g., non-ionic surfactant with anappropriate HLB number) may be employed. Furthermore, other types oftreatment are contemplated, such as antimicrobial photodynamic therapy,where enhanced or controlled delivery of a photosensitizer through anepithelium may be advantageous. Some microbes, such as fungi, havedormant phases, while other microbes, such as Acanthamoeba, can createcystic cell membrane barriers. Advantageously, additives that enhancepermeability can increase penetration and uptake of photosensitizer bymicrobes/pathogens and enhance the antimicrobial effect of thephotosensitizer. For instance, photosensitizer formulations employing anon-ionic permeability enhancer may be particularly effective forpenetrating cysts, ulcers, etc. and treating microbes/pathogens. Otheraspects of antimicrobial photodynamic therapy are described in U.S.patent application Ser. No. 15/137,748, filed Apr. 25, 2016 and titled“Systems and Methods for Photoactivating a Photosensitizer Applied to anEye,” the contents of which is incorporated entirely herein byreference.

When riboflavin absorbs radiant energy, especially light, it undergoesphoto activation. There are two photochemical kinetic pathways forriboflavin photoactivation, Type I and Type II. Some of the reactionsinvolved in both the Type I and Type II mechanisms are as follows:

Common Reactions:Rf→Rf ₁ *,I;  (r1)Rf ₁ *→Rf,κ1;  (r2)Rf ₁ *→Rf ₃*,κ2;  (r3)

Type I Reactions:Rf ₃ *+DH→RfH ^(•) +D ^(•),κ3;  (r4)2RfH ^(•) →Rf+RfH ₂,κ4;  (r5)

Type II Reactions:Rf ₃*+O₂ →Rf+O₂ ¹,κ5;  (r6)DH+O₂ ¹ →D _(ox),κ6;  (r7)D _(ox) +DH→D−D,κ7;CXL  (r8)

In the reactions described herein, Rf represents riboflavin in theground state. Rf*₁ represents riboflavin in the excited singlet state.Rf*₃ represents riboflavin in a triplet excited state. Rf^(•−) is thereduced radical anion form of riboflavin. RfH^(•) is the radical form ofriboflavin. RfH₂ is the reduced form of riboflavin. DH is the substrate.DH^(•+) is the intermediate radical cation. D^(•) is the radical. D_(ox)is the oxidized form of the substrate.

Riboflavin is excited into its triplet excited state Rf*₃ as shown inreactions (r1) to (r3). From the triplet excited state Rf*₃, theriboflavin reacts further, generally according to Type I or Type IImechanisms. In the Type I mechanism, the substrate reacts with theexcited state riboflavin to generate radicals or radical ions,respectively, by hydrogen atoms or electron transfer. In Type IImechanism, the excited state riboflavin reacts with oxygen to formsinglet molecular oxygen. The singlet molecular oxygen then acts ontissue to produce additional cross-linked bonds.

Oxygen concentration in the cornea is modulated by UVA irradiance andtemperature and quickly decreases at the beginning of UVA exposure.Utilizing pulsed light of a specific duty cycle, frequency, andirradiance, input from both Type I and Type II photochemical kineticmechanisms can be employed to achieve a greater amount of photochemicalefficiency. Moreover, utilizing pulsed light allows regulating the rateof reactions involving riboflavin. The rate of reactions may either beincreased or decreased, as needed, by regulating, one of the parameterssuch as the irradiance, the dose, the on/off duty cycle, riboflavinconcentration, soak time, and others. Moreover, additional ingredientsthat affect the reaction and cross-linking rates may be added to thecornea.

If UVA radiation is stopped shortly after oxygen depletion, oxygenconcentrations start to increase (replenish). Excess oxygen may bedetrimental in the conical cross-linking process because oxygen is ableto inhibit free radical photopolymerization reactions by interactingwith radical species to form chain-terminating peroxide molecules. Thepulse rate, irradiance, dose, and other parameters can be adjusted toachieve a more optimal oxygen regeneration rate. Calculating andadjusting the oxygen regeneration rate is another example of adjustingthe reaction parameters to achieve a desired amount of conicalstiffening.

Oxygen content may be depleted throughout the cornea, by variouschemical reactions, except for the very thin corneal layer where oxygendiffusion is able to keep up with the kinetics of the reactions. Thisdiffusion-controlled zone will gradually move deeper into the cornea asthe reaction ability of the substrate to uptake oxygen decreases.

Riboflavin is reduced (deactivated) reversibly or irreversibly and/orphoto-degraded to a greater extent as irradiance increases. Photonoptimization can be achieved by allowing reduced riboflavin to return toground state riboflavin in Type I reactions. The rate of return ofreduced riboflavin to ground state in Type I reactions is determined bya number of factors. These factors include, but are not limited to,on/off duty cycle of pulsed light treatment, pulse rate frequency,irradiance, and dose. Moreover, the riboflavin concentration, soak time,and addition of other agents, including oxidizers, affect the rate ofoxygen uptake. These and other parameters, including duty cycle, pulserate frequency, irradiance, and dose can be selected to achieve moreoptimal photon efficiency and make efficient use of both Type I as wellas Type II photochemical kinetic mechanisms for riboflavinphotosensitization. Moreover, these parameters can be selected in such away as to achieve a more optimal chemical amplification effect.

In addition to the photochemical kinetic reactions (r1)-(r8) above,however, the present inventors have identified the followingphotochemical kinetic reactions (r9)-(r26) that also occur duringriboflavin photoactivation:

Additional:

Aggregation:

Peroxides:2O₂ ⁻→O₂+H₂O₂,κ₁₂  (r25)OH^(∘) +CXL→inert products,κ_(OH)  (r26)

FIG. 2A illustrates a diagram for the photochemical kinetic reactionsprovided in reactions (r1) through (r26) above. The diagram summarizesphotochemical transformations of riboflavin (Rf) under UVAphotoactivating light and its interactions with various donors (DH) viaelectron transfer. As shown, cross-linking activity occurs: (A) throughthe presence of singlet oxygen in reactions (r6) through (r8) (Type IImechanism); (B) without using oxygen in reactions (r4) and (r17) (Type Imechanism); and (C) through the presence of peroxide (H₂O₂), superoxide(O₂ ⁻), and hydroxyl radicals (.OH) in reactions (r13) through (r17).

As shown in FIG. 2A, the present inventors have also determined that thecross-linking activity is generated to a greater degree from reactionsinvolving peroxide, superoxide, and hydroxyl radicals. Cross-linkingactivity is generated to a lesser degree from reactions involvingsinglet oxygen and from non-oxygen reactions. Some models based on thereactions (r1)-(r26) may account for the level of cross-linking activitygenerated by the respective reactions. For instance, where singletoxygen plays a smaller role in generating cross-linking activity, modelsmay be simplified by treating the cross-linking activity resulting fromsinglet oxygen as a constant.

All the reactions start from Rf₃* as provided in reactions (r1)-(r3).The quenching of Rf₃* occurs through chemical reaction with ground stateRf in reaction (r10), and through deactivation by the interaction withwater in reaction (r9).

As described above, excess oxygen may be detrimental in cornealcross-linking process. As shown in FIG. 2A, when the system becomesphoton-limited and oxygen-abundant, cross-links can be broken fromfurther reactions involving superoxide, peroxide, and hydroxyl radicals.Indeed, in some cases, excess oxygen may result in net destruction ofcross-links versus generation of cross-links.

As described above, a large variety of factors affect the rate of thecross-linking reaction and the amount of biomechanical stiffnessachieved due to cross-linking. A number of these factors areinterrelated, such that changing one factor may have an unexpectedeffect on another factor. However, a more comprehensive model forunderstanding the relationship between different factors forcross-linking treatment is provided by the photochemical kineticreactions (r1)-(r26) identified above. Accordingly, systems and methodscan adjust various parameters for cross-linking treatment according tothis photochemical kinetic cross-linking model, which provides a unifieddescription of oxygen dynamics and cross-linking activity. The model canbe employed to evaluate expected outcomes based on differentcombinations of treatment parameters and to identify the combination oftreatment parameters that provides the desired result. The parameters,for example, may include, but is not limited to: the concentration(s)and/or soak times of the applied cross-linking agent; the dose(s),wavelength(s), irradiance(s), duration(s), and/or on/off duty cycle(s)of the photoactivating light; the oxygenation conditions in the tissue;and/or presence of additional agents and solutions.

A model based on the reactions (r1)-(r19) has been validated by at leastsix different methods of evaluating cross-linking activity:

-   -   Extensiometry experiments    -   Oxygen depletion experiments    -   Non-linear optical microscopy fluorescence experiments    -   Fluorescence data based on papain digestion method experiments    -   Brillouin microscopy experiments    -   Corneal stromal demarcation line correlation experiments

For extensiometry experiments, corneas were soaked with riboflavin for20 minutes and exposed to UVA photoactivating light in ambient air at anirradiance of 3 mW/cm² for 7.5 minutes (dose of 1.35 J/cm²), 15 minutes(dose of 2.70 J/cm²), 30 minutes (dose of 5.4 J/cm²), 45 minutes (doseof 8.10 J/cm²), 120 minutes (dose of 21.6 J/cm²), and 150 minutes (doseof 27.0 J/cm²). Extensiometry measurements were taken for 200 μm flapsof the corneas. FIG. 31 illustrates a graph of cross-link profiles(cross-link concentration as a function of corneal depth) calculated bythe model for each cross-linking treatment. FIG. 32 illustrates acorrelation between the extensiometry measurements and the valuescalculated by the model (area under the curve for 200 μm). In general,there is a good correlation between the biomechanics determined in theextensiometry experiments and the values calculated by the model. It canalso be seen from FIG. 31 that cross-linking can saturate whenparticular treatment parameters (e.g., longer irradiation times) areemployed.

For the oxygen depletion experiments, O₂ concentrations were measuredand calculated at a depth of approximately 100 μm to approximately 200μm for corneas treated with riboflavin. FIG. 3A illustrates a graph ofdata showing the correlation between the theoretical values based on themodel and experimental data for corneas exposed to continuous wave UVAphotoactivating light at an irradiance of 3 mW/cm². FIGS. 3B-Cillustrate graphs of data showing the correlation between model valuesand experimental data for corneas exposed to long term pulses and shortterm pulses, respectively, at an irradiance of 3 mW/cm².

For the non-linear optical microscopy fluorescence experiments, thecross-linking profiles based on corneal depth were determined forcorneas treated with riboflavin and exposed to UVA photoactivating lightat an irradiance of 3 mW/cm². FIG. 4 illustrates a graph of data showingthe correlation between model and experimental data for corneas exposedfor 15 minutes and 30 minutes. The third party experimental data waspublished in Dongyul Chai et al. “Quantitative Assessment ofUVA-riboflavin Corneal Cross-Linking Using Nonlinear OpticalMicroscopy.” Investigative Ophthalmology & Visual Science. June 2011,Vol. 52, No. 7, pp. 4231-4238, the contents of which are incorporatedentirely herein by reference.

For the fluorescence data based on papain digestion method experiments,cross-linking concentrations were evaluated based on fluorescent lightintensity. FIG. 5A illustrates a graph of data showing the correlationof model values and experimental data for corneal flaps (taken from 0 toapproximately 100 μm deep) exposed to combinations of riboflavinconcentrations (0.1%, 0.25%, and 0.5%) and 5.4 J/cm² doses of UVAphotoactivating light at irradiances of 3 mW/cm² and 30 mW/cm² for 3minutes and 30 minutes. Similarly, FIG. 5B illustrates a graph of datashowing the correlation of model values and experimental data forcorneal flaps (taken from approximately 100 μm to approximately 200 μmdeep) exposed to combinations of riboflavin concentrations (0.1%, 0.25%,and 0.5%) and 5.4 J/cm² doses of UVA photoactivating light atirradiances of 3 mW/cm² and 30 mW/cm² for 3 minutes and 30 minutes. FIG.5C illustrates a graph of data showing the correlation of model valuesand experimental data for corneal flaps treated with a concentration ofriboflavin and exposed to full oxygen concentration and 5.4 J/cm² and7.2 J/cm² doses of continuous wave UVA photoactivating light atirradiances of 3 mW/cm², 10 mW/cm², 15 mW/cm², 30 mW/cm², 45 mW/cm², 60mW/cm², and 100 mW/cm².

FIG. 5D illustrates a graph of data showing the correlation of modelvalues and experimental data for corneal flaps (taken from approximately0 μm to approximately 200 μm deep) treated with a concentration of 0.1%riboflavin and exposed to air or full oxygen concentration and a 5.4J/cm² doses of continuous wave UVA photoactivating light at irradiancesof 3 mW/cm², 10 mW/cm², 15 mW/cm², 30 mW/cm², 45 mW/cm², 60 mW/cm², and100 mW/cm². The values at irradiance 3 mW/cm² under 100% oxygen showsthe effect of quenching Rf₃* by oxygen.

Brillouin microscopy experiments are described in Scarcelli G et al.,Invest. Ophthalmol. Vis. Sci. (2013), 54: 1418-1425, the contents ofwhich are incorporated entirely herein by reference. The experimentsmeasured Brillouin modulus values quantifying corneal mechanicalproperties after various cross-linking treatments. FIG. 28A showsBrillouin modulus values measured at anterior, central, and posteriorsections of corneas experimentally soaked in riboflavin for variousdurations and irradiated with UV light for various durations. FIG. 28Billustrates the correlation between the experimentally measured valuesand values calculated with the model for various treatments. Treatment Ain FIG. 28B corresponds to a soak time of 30 minutes and irradiation of5 minutes. Treatment B corresponds to a soak time of 30 minutes andirradiation of 15 minutes. Treatment C corresponds to a soak time of 30minutes and irradiation of 30 minutes. Treatment D corresponds to a soaktime of 30 minutes and irradiation of 5 minutes. Treatment E correspondsto a soak time of 5 minutes and irradiation of 30 minutes. Treatment Ecorresponds to a soak time of 30 minutes and irradiation of 30 minutes.

For the corneal stromal demarcation correlation experiments, cornealstromal demarcation lines were evaluated for treated corneas. A methodfor these experiments involves slit-lamp examination (slit projectionand Scheimpflug camera). See Theo Seiler and Farhad Hafezi, “CornealCross-Linking-Induced Stromal Demarcation Line,” Cornea, October 2006;25:1057-59, the contents of which are incorporated entirely herein byreference. Another method involves corneal optical coherence tomography(OCT). See Luigi Fontana, Antonello Moramarco, “Esperienze personali conCXL accelerate,” UOC Oculistica ASMN-IRCCS Reggio Emilia. Roma, 20settembre 2014, the contents of which are incorporated entirely hereinby reference. A further method involves confocal microscopy. See C.Mazzotta et al., “Treatment of Progressive Keratoconus by of CornealCollagen In Vivo Confocal Microscopy in Humans,” Cornea, vol. 26, no. 4,May 2007, the contents of which are incorporated entirely herein byreference.

Corneal stromal healing involves the deposition of new collagen, whichproduces haze and scattering. Slit-lamp examination and OCT can detectthis hyper-reflectivity and possibly a significant change in the spatialorder factor (change in birefringence, i.e., index variation). Cornealstromal demarcation lines indicate the threshold at which the healingresponse occurs. This conclusion is corroborated by confocal microscopy.The demarcation lines can also be seen as a transition zone betweencross-linked anterior corneal stroma and untreated posterior cornealstroma.

The six evaluations described above show a strong correlation betweenthe experimental data and the calculations generated by a model based onthe photochemical kinetic reactions identified above. The model isextremely effective and accurate in predicting the results of riboflavincross-linking treatments applied according to various combinations ofparameters. Accordingly, using such a model, systems and methods canmore efficiently and predictably achieve a desired profile ofcross-linking activity throughout the cornea. The model allows thesystems and methods to identify a more optimal combination of parametersfor cross-linking treatment. Therefore, the model can be used todetermine the set up for different aspects of cross-linking treatmentsystems as described above.

As shown in FIG. 2B, aspects of the system of reactions can be affectedby different parameters. For instance, the irradiance at whichphotoactivating light is delivered to the system affects the photonsavailable in the system to generate Rf₃* for subsequent reactions.Additionally, delivering greater oxygen into the system drives theoxygen-based reactions. Meanwhile, pulsing the photoactivating lightaffects the ability of the reduced riboflavin to return to ground stateriboflavin by allowing additional time for oxygen diffusion. Of course,other parameters can be varied to control the system of reactions.

A model based on the photochemical kinetic reactions (r1)-(r26) cangenerate cross-link profiles for treatments using different protocols asshown in FIGS. 7A-C. In particular, each protocol determines the dose ofthe photoactivating UVA light, the irradiance for the UVAphotoactivating light, the treatment time, and the concentration ofoxygen delivered to the corneal surface. The cornea has been treatedwith a formulation including 0.1% concentration riboflavin. FIG. 7Aillustrates cross-link profiles for treatments that deliver a dose of7.2 J/cm² of UVA light under normal (ambient) oxygen according todifferent irradiances and different treatment times. FIG. 7B illustratescross-link profiles for treatments that employ different irradiances ofcontinuous or modulated (pulsed) UVA light and different treatment timesunder normal or 100% oxygen concentration. FIG. 7C illustratescross-link profiles for treatments that deliver an irradiance of 3 mW ofUVA light for 30 minutes with different oxygen conditions (normal, 100%,or 0.01×) at the corneal surface.

The cross-link profiles in FIGS. 7A-C provide the cross-linkconcentration as a function of corneal depth. In general, thethree-dimensional distribution of cross-links in the cornea as indicatedby each cross-link profile depends on the combination of differenttreatment parameters. Protocols employing different sets of treatmentparameters can be provided as input into the model and the model canoutput the resulting three-dimensional distribution of cross-links inthe cornea. Accordingly, the model can be used to select treatmentparameters to achieve the desired distribution of cross-links in thecornea.

FIG. 6A illustrates a graph of data showing the correlation of modelvalues and experimental data for the depths of corneal stromaldemarcation lines for the protocols described in FIG. 6B.

As described above, corneal stromal demarcation lines indicate thetransition zone between cross-linked anterior corneal stroma anduntreated posterior corneal stroma. As also shown in FIG. 8A-C,cross-link profiles generated by the model can be evaluated to determinethe depth at which the demarcation line may appear at a cross-linkconcentration of approximately 5 mol/m³. Here, the demarcation line maybe understood as the threshold at which a healing response occurs inresponse to the distribution of cross-links as well as the effect ofreactive oxygen species on the corneal tissue. The cornea has beentreated with a formulation including 0.1% concentration riboflavin. FIG.8A illustrates a cross-link profile for a treatment that delivers a doseof 5.4 J/cm² of photoactivating UVA light under normal oxygen accordingto an irradiance of 3 mW/cm² and a treatment time of 30 minutes. FIG. 8Ashows that a cross-link concentration of approximately 5 mol/m³(demarcation line) occurs at a depth of approximately 290 μm in theresulting cross-link profile. FIG. 8B illustrates cross-link profilesfor treatments that deliver different doses of photoactivating UVA lightaccording to different irradiances and different treatment times undernormal oxygen. FIG. 8C illustrates cross-link profiles for treatmentsthat deliver different doses of photoactivating UVA light according todifferent irradiances and different treatment times under normal or 100%oxygen concentration.

FIGS. 8B-C shows that the depths for the demarcation line vary with thedifferent cross-link profiles generated by the different sets oftreatment parameters. The depths of the demarcation line indicated bythe different cross-link profiles may be employed to select treatmentparameters. For instance, treatment parameters may be selected to ensurethat the cross-links do not occur at a depth where undesired damage mayresult to the endothelium. This analysis allows the treatment system toaccommodate different corneal thicknesses, particularly thin corneas.

Correspondingly, FIGS. 9A-B illustrate graphs of demarcation depth(cross-link concentration of approximately 5 mol/m³) as a function ofdose of UVA photoactivating light. The determination of the demarcationdepths are based on cross-link profiles generated by the model fortreatments using different protocols. The cornea has been treated with aformulation including 0.1% concentration riboflavin. FIG. 9A illustratesgraphs for treatments that deliver continuous or pulsed UVAphotoactivating light according to different irradiances under normaloxygen. FIG. 9B illustrates graphs for treatments that delivercontinuous or pulsed UVA photoactivating light according to differentirradiances under a greater concentration of oxygen.

FIG. 10 illustrates the cross-link profiles for treatments employingdifferent protocols as generated by the model. FIG. 10 also shows ademarcation line that corresponds to biomechanical stiffness thresholdat a cross-link concentration of 10 mol/m³. The demarcation lineintersects the cross-link profiles at varying depths (biomechanicalstiffness depth) based on the different treatment parameters of theprotocols. FIG. 11 illustrates the measurement of maximum keratometry(K_(max)) (diopters) at three, six, and twelve months relative to abaseline for corneas that were experimentally treated according to theprotocols employed for FIG. 10.

FIGS. 12A-B illustrate the correlation between the experimental data ofFIG. 11 and the cross-link profiles generated for FIG. 10 by the model.For the biomechanical stiffness depth determined for each protocol inFIG. 10, FIG. 12A plots the experimental change of K_(max) for monthssix and twelve corresponding to the respective protocol. FIG. 12A alsoshows a quadratic fit of the plotted data for each month six and twelve.The quadratic fit is consistent with the quadratic nature of shearforces (in the x-y plane) resulting from a force placed on a disk (alongthe z-axis) according to thin shell theory.

Meanwhile, for the area above the demarcation line for the cross-linkprofile for each protocol in FIG. 10, FIG. 12B plots the experimentalchange of K_(max) for months six and twelve corresponding to therespective protocol. FIG. 12B also shows a linear fit of the plotteddata for each month six and twelve.

The quadratic fit for the two curves in FIG. 12A are substantiallysimilar. Similarly, the linear fit for the two curves in FIG. 12B aresubstantially similar. The correlations shown in FIGS. 12A-B indicatethat there is a predictable biomechanical/healing response over time fora given set of treatment parameters. In view of the verification of theexperimental data points, the model, as well as thin shell analysis, onecan predictably determine refractive change according to the radius anddepth of the disk corresponding to the myopic correction. In general,the distribution of cross-links effects refractive change. By accuratelydetermining the distribution of cross-links, the model can be employedto determine this refractive change.

FIG. 29 illustrates concentration of cross-link concentration as well asa function of corneal depth with a demarcation depth of approximately360 μm. FIG. 29 also shows a line of demarcation analysis using a secondderivative and threshold algorithm. The second derivative can also becalculated as shown. At the illustrated demarcation depth, the secondderivative has a threshold derivative of 180 mM/mm².

FIGS. 35-36 illustrate graphs of cross-link profiles for treatmentsemploying different protocols, as generated by the model. The graphsalso show a potential threshold cross-link concentration ofapproximately 4.4 mM/m³ for the demarcation line. As shown, thedemarcation depth is taken at a shift of approximately 125 μm from theintersection of the cross-link profile curve and the potential thresholdcross-link concentration. In FIG. 35, for instance, the demarcation lineoccurs at a depth of approximately 350 μm.

The model of photochemical kinetic reactions was further evaluatedagainst the results reported by fourteen different studies in total.FIG. 30 illustrates a graph of data showing the correlation of modelvalues and experimental data for the depths of corneal stromaldemarcation lines for protocols described in these fourteen separatestudies. The measurements are taken from the epithelial surface. Thestandard deviation is approximately 20 μm.

TABLE 1 shows further shows data for determination of demarcation linedepth for different treatments.

TABLE 2 Demarcation line depth Conventional CXL C-light ACXL P-lightACXL TE CXL TE ACXL CXL Treatments 44 eyes 10 eyes 10 eyes 10 eyes 10eyes 84 eyes 3 mW 30 mW 30 mW 3 mW 45 mW Average demarcation 350 ± 20 μm200 ± 20 μm 250 ± 20 μm 100 ± 20 μm 100 ± 20 μm line depth (measuredfrom epithelial surface) *Average epithelial thickness: 50 ± 10 μm. CXL,conventional crosslinking: C-light ACXL, continous light acceleratedcrosslinking: P-light ACXL, pulsed light accelerated crosslinking; TECXL, transepithelial crosslinking; TE ACXL, transepithelial acceleratedcrosslinking.

The reported standard deviations reveal variability in the depth of thedemarcation line for nominally equivalent clinical protocols. Suchvariability may be the result of aspects of measurement error, clinicaltechnique, protocol, the riboflavin formulation, and/or the equipmentemployed. An analysis of the reported variability was performed with themodel of photochemical kinetic reactions. FIG. 37 illustrates that thedemarcation line depth may be affected by aspects of the riboflavinconcentration, the use of thickening agent, irradiation (UVA) devicecalibration, irradiation (UVA) beam profile, and/or geographic factors.

In sum, the line of demarcation is predicted accurately the model ofphotochemical kinetic reactions. As such, the model of photochemicalkinetic reactions may be used to treat corneas, particularly thinnercorneas, more safely. However, changes in clinical protocol may resultin variability in the depth of the line of demarcation line andpotentially clinical outcomes, suggesting the importance of precision incross-linking treatment methodology. Consistency in protocol, techniqueand equipment can enhance the predictability of the clinical outcomes.

According to an embodiment, FIG. 13 illustrates the example system 100employing a model based on the photochemical kinetic reactions(r1)-(r26) identified above to determine an amount of cross-linking thatresults from treatment parameters and/or other related information. Thecontroller 120 includes a processor 122 and computer-readable storagemedia 124. The storage media 124 stores program instructions fordetermining an amount of cross-linking when the photoactivating lightfrom the light source 110 is delivered to a selected region of a corneatreated with a cross-linking agent. In particular, a photochemicalkinetic model 126 based on the reactions (r1)-(r26) may include a firstset of program instructions A for determining cross-linking resultingfrom reactions involving reactive oxygen species (ROS) includingcombinations of peroxides, superoxides, hydroxyl radicals, and/orsinglet oxygen and a second set of program instructions B fordetermining cross-linking from reactions not involving oxygen. Thecontroller 120 receives input relating to treatment parameters and/orother related information. The controller 120 can then execute theprogram instructions A and B to output information relating tothree-dimensional cross-link distribution(s) for the selected region ofthe cornea based on the input. The three-dimensional cross-linkdistribution(s) may then be employed to determine how to control aspectsof the light source 110, the optical elements 112, the cross-linkingagent 130, the applicator 132, the oxygen source 140, and/or oxygendelivery device 142 in order to achieve a desired treatment in selectedregion of the cornea. (Of course, the system 100 shown in FIG. 13 andthis process can be used for treatment of more than one selected regionof the same cornea.)

According to one implementation, the three-dimensional cross-linkdistribution(s) may be evaluated to calculate a threshold depthcorresponding to a healing response due to the cross-links and an effectof the reactive-oxygen species in the selected region of the cornea.Additionally or alternatively, the three-dimensional cross-linkdistribution(s) may be evaluated to calculate a biomechanical tissuestiffness threshold depth corresponding to a biomechanical tissueresponse in the selected region of the cornea. The information on thedepth of the healing response and/or the biomechanical tissue stiffnessin the cornea can be employed to determine how to control aspects of thelight source 110, the optical elements 112, the cross-linking agent 130,the applicator 132, the oxygen source 140, and/or oxygen delivery device142. Certain healing response and/or biomechanical tissue stiffness maybe desired or not desired at certain depths of the cornea.

Referring to FIG. 33, the photochemical kinetic model allows particularaspects of the photochemical process to be controlled or otherwiseinfluenced to produce desired cross-linking activity. For instance,different additives, such as iron, may be employed to affect mechanismsat different points of the photochemical process as shown in FIG. 33.FIG. 34 shows the effect of the various additives in FIG. 33 oncross-linking activity.

According to another embodiment, FIG. 38 illustrates the example system100 employing the photochemical kinetic model 126 to determine treatmentparameters for achieving desired biomechanical changes in the cornea,e.g., a refractive correction. As in FIG. 13, the controller 120includes the processor 122 and the computer-readable storage media 124.In the example of FIG. 14, however, the storage media 124 stores programinstructions 125 for determining what treatment parameters may beemployed to achieve desired biomechanical changes. The programinstructions 125 are based on the photochemical kinetic model 126 whichemploy the reactions (r1)-(r26) to determine cross-linking resultingfrom (i) reactions involving reactive oxygen species (ROS) includingcombinations of peroxides, superoxides, hydroxyl radicals, and/orsinglet oxygen and (ii) reactions not involving oxygen.

Using the photochemical kinetic model 126, a three-dimensionaldistribution of resulting cross-links throughout the treated cornealtissue can be determined for a combination of treatment parameters. Asdescribed above, parameters for cross-linking treatment may include: theconcentration(s) and/or soak times of the applied cross-linking agent;the dose(s), wavelength(s), irradiance(s), duration(s), on/off dutycycle(s), and/or other illumination parameters for the photoactivatinglight; the oxygenation conditions in the tissue; and/or presence ofadditional agents and solutions. The resulting distribution ofcross-links determined from the photochemical kinetic model 126 can becorrelated to a particular biomechanical change in the cornea. FIGS.12A-B show, for instance, the correlation between the distribution ofcross-links and refractive change.

As shown in FIG. 38, the controller 120 receives an input 12 relating tothe initial biomechanical state of the cornea and an input 14 indicatinga desired biomechanical change for the cornea, e.g., for refractivecorrection. The initial biomechanical state, for instance, can bedetermined according to approaches described in U.S. Patent ApplicationPublication No. 2012/0215155 referenced above. In some cases, the input12 may be provided by a measurement system communicatively coupled tothe controller 120. It is understood that the initial biomechanicalstate may reflect the state of the cornea prior to any treatment orduring a treatment.

The inputs 12, 14 may be expressed in terms of corneal topography (i.e.,shape), corneal strength (i.e., stiffness), and/or corneal thickness.For instance, the desired biomechanical change for refractive correctionmay be determined from a correction specified (by a practitioner) indiopters, e.g., “a 1.5 diopter correction.”

A desired biomechanical change in the cornea can be correlated to aparticular distribution of cross-links as determined by thephotochemical kinetic model 126. As such, the controller 120 can executethe program instructions 125 to determine the particular distribution ofcross-links 16 that can generate the desired biomechanical changespecified by the input 14 in a cornea having the initial biomechanicalstate specified by the input 12. After determining the distribution ofcross-links 16 for the desired biomechanical change, the controller 120can prescribe a set of treatment parameters for achieving the specifieddistribution of cross-links.

As the studies above establish, however, the distribution of cross-links16 might be achieved in many cases by more than one set of treatmentparameters. For instance, depending on the photochemical kineticreactions, similar distributions of cross-links may be achieved byapplying: (i) a lower dose of photoactivating light for a longer amountof time, or (ii) a higher dose of photoactivating light for a shorteramount of time. Therefore, more than one set of treatment parameters 18for achieving the distribution of cross-links 16 may be identified.

With more than one possible set of treatment parameters 18, apractitioner can optimize the treatment for certain preferredparameters, such as treatment time or dose of photoactivating light. Forinstance, the practitioner may optimize the treatment parameters toachieve shorter treatment times. For this preference, the controller 120may prescribe a set of illumination parameters that provide a largerdose of photoactivating light that yields the distribution ofcross-links 16 over shorter illumination durations. Conversely, thepractitioner may optimize the treatment parameters to employ smallerdoses of photoactivating light. For this second preference, thecontroller 120 may prescribe a set of illumination parameters thatprovide a smaller dose of photoactivating light that yields thedistribution of cross-links 16 over longer illumination durations.

In general, to achieve the distribution of cross-links 16, thecontroller 120 may identify any of the different combinations 18 ofvalues for a set of treatment parameters A, B, C, D, E, etc., asdescribed above. The practitioner can set preferences for one or more ofthese treatment parameters. For instance, the practitioner may initiallyset a preferred value or range of preferred values for parameter A. Inresponse, the controller 120 can specify combinations of values for theremaining parameters B, C, D, E, etc., that meet the preference forparameter A while achieving the distribution of cross-links 16. Thepractitioner may make selections for the values of the parameters B, C,D, and/or E, etc., based on further preferences to arrive at anoptimized set of treatment parameters 18 a. The process of optimizingthe treatment parameters may be iterative as the values for thetreatment parameters are incrementally tuned to meet preferences havingvarying priorities.

In some embodiments, the practitioner may manage the optimizationprocess through a series of selections and other inputs via a userinterface (not shown) coupled to the controller 120. In some cases, theinputs 12, 14 may also be provided through such a user interface.

The final set of treatment parameters 18 a can then be employed todetermine how to control aspects of the light source 110, the opticalelements 112, the cross-linking agent 130, the applicator 132, theoxygen source 140, oxygen delivery device 142, etc., in order to achievea desired treatment in selected region of the cornea.

Correspondingly, FIG. 39 illustrates an example method 200 for employinga model of photochemical kinetic reactions (r1)-(r26) to determinetreatment parameters for achieving desired biomechanical changes. Instep 202, information relating to the initial biomechanical state of acornea is received. In step 204, information relating to a desiredbiomechanical change for the cornea, e.g., for refractive correction, isreceived. In step 206, a distribution of cross-links is determined toachieve the desired biomechanical change in a cornea having the initialbiomechanical state. In step 208, one or more sets of treatmentparameters are determined to achieve the distribution of cross-links. Inassociation with step 208, one or more preferences for treatmentparameters may be received in step 210, and the treatment parameters maybe optimized in step 212 based on the one or more preferences todetermine a final set of treatment parameters that can be implemented ina treatment system (e.g., the example system 100) to achieve thedistribution of cross-links.

As described above, according to some aspects of the present disclosure,some or all of the steps of the above-described and illustratedprocedures can be automated or guided under the control of a controller(e.g., the controller 120). Generally, the controllers may beimplemented as a combination of hardware and software elements. Thehardware aspects may include combinations of operatively coupledhardware components including microprocessors, logical circuitry,communication/networking ports, digital filters, memory, or logicalcircuitry. The controller may be adapted to perform operations specifiedby a computer-executable code, which may be stored on a computerreadable medium.

As described above, the controller may be a programmable processingdevice, such as an external conventional computer or an on-board fieldprogrammable gate array (FPGA) or digital signal processor (DSP), thatexecutes software, or stored instructions. In general, physicalprocessors and/or machines employed by embodiments of the presentdisclosure for any processing or evaluation may include one or morenetworked or non-networked general purpose computer systems,microprocessors, field programmable gate arrays (FPGA's), digital signalprocessors (DSP's), micro-controllers, and the like, programmedaccording to the teachings of the example embodiments of the presentdisclosure, as is appreciated by those skilled in the computer andsoftware arts. The physical processors and/or machines may be externallynetworked with the image capture device(s), or may be integrated toreside within the image capture device. Appropriate software can bereadily prepared by programmers of ordinary skill based on the teachingsof the example embodiments, as is appreciated by those skilled in thesoftware art. In addition, the devices and subsystems of the exampleembodiments can be implemented by the preparation ofapplication-specific integrated circuits or by interconnecting anappropriate network of conventional component circuits, as isappreciated by those skilled in the electrical art(s). Thus, the exampleembodiments are not limited to any specific combination of hardwarecircuitry and/or software.

Stored on any one or on a combination of computer readable media, theexample embodiments of the present disclosure may include software forcontrolling the devices and subsystems of the example embodiments, fordriving the devices and subsystems of the example embodiments, forenabling the devices and subsystems of the example embodiments tointeract with a human user, and the like. Such software can include, butis not limited to, device drivers, firmware, operating systems,development tools, applications software, and the like. Such computerreadable media further can include the computer program product of anembodiment of the present disclosure for performing all or a portion (ifprocessing is distributed) of the processing performed inimplementations. Computer code devices of the example embodiments of thepresent disclosure can include any suitable interpretable or executablecode mechanism, including but not limited to scripts, interpretableprograms, dynamic link libraries (DLLs), Java classes and applets,complete executable programs, and the like. Moreover, parts of theprocessing of the example embodiments of the present disclosure can bedistributed for better performance, reliability, cost, and the like.

Common forms of computer-readable media may include, for example, afloppy disk, a flexible disk, hard disk, magnetic tape, any othersuitable magnetic medium, a CD-ROM, CDRW, DVD, any other suitableoptical medium, punch cards, paper tape, optical mark sheets, any othersuitable physical medium with patterns of holes or other opticallyrecognizable indicia, a RAM, a PROM, an EPROM, a FLASH-EPROM, any othersuitable memory chip or cartridge, a carrier wave or any other suitablemedium from which a computer can read.

While the present disclosure has been described with reference to one ormore particular embodiments, those skilled in the art will recognizethat many changes may be made thereto without departing from the spiritand scope of the present disclosure. Each of these embodiments andobvious variations thereof is contemplated as falling within the spiritand scope of the invention. It is also contemplated that additionalembodiments according to aspects of the present disclosure may combineany number of features from any of the embodiments described herein.

What is claimed is:
 1. A formulation for an eye treatment, comprising: aphotosensitizer solution including a photosensitizer that, in responseto absorbing photoactivating illumination, produces reactive radicalsthat interact with corneal tissue to generate cross-linking activity inthe corneal tissue, the photosensitizer having molecules that are largerelative to tight junctions of an epithelium of the corneal tissue; anda permeability enhancing composition including at least one of IGEPALCO-630, Triton X-100, Polidocanol, or IGEPAL CO-720, wherein thephotosensitizer solution includes a concentration of at least 0.1%riboflavin.
 2. The formulation of claim 1, further comprising at leastone additive selected from the group consisting of iron, copper,manganese, chromium, vanadium, aluminum, cobalt, mercury, cadmium,nickel, arsenic, 2,3-butanedione, and folic acid.
 3. The formulation ofclaim 1, further comprising at least one additive including iron.
 4. Amethod for treating an eye, comprising: applying a formulation tocorneal tissue, the formulation including: a photosensitizer solutionincluding a photosensitizer that, in response to absorbingphotoactivating illumination, produces reactive radicals that interactwith the corneal tissue to generate cross-linking activity in thecorneal tissue, the photosensitizer having molecules that are largerelative to tight junctions of an epithelium of the corneal tissue; anda permeability enhancing composition including at least one of IGEPALCO-630, Triton X-100, Polidocanol, or IGEPAL CO-720; and photoactivatingthe photosensitizer by delivering a dose of illumination to the cornealtissue, wherein the photosensitizer solution includes a concentration ofat least 0.1% riboflavin.
 5. The method of claim 4, wherein theformulation further includes at least one additive selected from thegroup consisting of iron, copper, manganese, chromium, vanadium,aluminum, cobalt, mercury, cadmium, nickel, arsenic, 2,3-butanedione,and folic acid.
 6. The method of claim 4, further comprising at leastone additive including iron.
 7. The formulation of claim 1, wherein thephotoactivating illumination is ultraviolet light, and in response toabsorbing the ultraviolet light, the photosensitizer undergoes areaction with oxygen to produce at least reactive oxygen species thatinteract with corneal collagen fibrils to induce covalent bonds thatbind together amino acids of the collagen fibrils, thereby crosslinkingthe fibrils.
 8. The method of claim 4, wherein the photoactivatingillumination is ultraviolet light, and in response to absorbing theultraviolet light, the photosensitizer undergoes a reaction with oxygento produce at least reactive oxygen species that interact with cornealcollagen fibrils to induce covalent bonds that bind together amino acidsof the collagen fibrils, thereby crosslinking the fibrils.